Construction of Highly Viable, Excitable, Aligned Human Induced Pluripotent Stem Cell-Derived Retinal Ganglion Cell Sheets on Poly-ε-caprolactone Nanofibers by Targeting Integrin β1-Mediated Adhesion Pathways

Abstract

Transplantation of induced pluripotent stem cell-derived retinal ganglion cells (RGCs) is a promising strategy for optic nerve regeneration and vision restoration in glaucoma. However, injections of conventional single-cell suspensions have low engraftment rates, hindering the formation of the complex neural circuits essential for functional recovery. This study addresses this challenge by investigating an in vitro biomimetic tissue engineering approach designed for future translational objectives, such as RGC transplantation therapy. We systematically evaluated coating materials and culture supplements critical for the survival and morphological development of RGCs on biocompatible, aligned poly-ε-caprolactone (PCL) nanofiber membranes. Notably, we identified the xeno-free extracellular matrix (ECM) conditions that promote RGC neurite extension at levels comparable to animal-derived ECM, thereby overcoming a major barrier to clinical translation of this technique. Furthermore, while RGCs typically have compromised viability and limited outgrowth on nanofibers, enhancing cell-scaffold adhesion signaling via the integrin β1/focal adhesion kinase pathway significantly improved both survival and neurite development. Crucially, RGCs on aligned PCL nanofibers extended neurites along the longitudinal axes of the nanofibers, with both somata and neurites exhibiting functional calcium responses to depolarization. These findings represent a significant advancement in biomimetic in vitro tissue engineering aimed at recapitulating retinal neurite orientation, providing a scalable platform for future nanofiber-based neural regeneration therapies, with the ultimate goal of clinical applicability.